Full text of "USPTO Patents Application 10687850"

See other formats

PCT

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :

G01N 27/327, 27/333, 33/50, 33/574,
33/68, 33/96

(11) International Publication Number: WO 97/29366

(43) Internationa] Publication Date: 1 4 August 1997 (1 4.08.97)

(21) International Application Number: PCT/AU97/0007 1

(22) International Filing Date: 10 February 1997 (10.02.97)

(30) Priority Data:
60/011,314

8 February 1996 (08.02.96) US

(71) Applicants (for all designated Stales except US): AUS-

TRALIAN MEMBRANE AND BIOTECHNOLOGY
RESEARCH INSTTTUTO [AU/AU]; 126 Grevffle Street
Chatswood, NSW 2067 (AU). THE UNIVERSITY OF
SYDNEY [AU/AU]; Sydney, NSW 2006 (AU).

(72) Inventors; and

(75) Inventors/Applicants (for US only): BRAACH-MAKSVYTIS,
Vijoleta, Lucija, Bronislava [AU/AU); 9 Darley Street, Dul-
wich Hills, NSW 2203 (AU). CORNELL, Bruce, Andrew
[AU/AU]; 58 Wycombe Road, Neutral Bay, NSW 2089
(AU). THOMSON, David, Geoffrey [AU/AU]; 19 Konin-
derie Parade, Narara, NSW 2250 (AU). RAGUSE, Burkbard
[DE/AU]; 2 Mudies Road, St. Ives, NSW 2075 (AU).

(74) Agent: F3. RICE & CO.; 28A Montague Street, Balmain,
NSW 2041 (AU).

(SI) Designated States: AL. AM, AT, AU, AZ, BA. BB, BG, BR,
BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE,
HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS,
LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL,
PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA,
UG, US, UZ, VN, YU, ARIPO patent (KE, LS, MW, SD,
SZ, UG), Eurasian patent (AM. AZ, BY, KG, KZ. MD, RU.
TJ. TM), European patent (AT, BE, CH, DE, DK, ES, FI,
FR. GB, GR. IE, IT, LU. MC, NL, PT, SE), OAPl patent
(BF, BJ, CF, CG, CI, CM, GA. GN, ML, MR, NE, SN. TD,
TG).

Published

With international search report.

(54) Title: ENZYME DETECTION BIOSENSORS
(57) Abstract

The present invention provides a biosensor for use in detecting the presence of an enzyme or enzymes in a sample. The biosensor
comprises a membrane and means for determining the impedance of the membrane. The membrane includes ionophores therein to which
are attached linkers. The linkers are cleavable by the enzyme or enzymes to be detected, with the cleavage of the linker causing a change
in the ability of ions to pass through the membrane via the ionophores.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under the PCT.

Unned Kingdom

Austria

Georgia

Australia

Guinea

Barbados

Greece

Be (gram

Hungary

Bark mi Faso

belaud

Bulgaria

Italy

Benin

Japan

Brazil

Kenya

Belarus

Kyrgyatan

Canada

l^mocratic People's Republic

Central African Republic

of Korea

Congo

Republic of Korea
Kazakhstan

Switzerland

Core dlvotre

Cameroon

Sri Lanka

China

Liberia

Czechoslovakia

Lithuania

Czech Republic

Luxembourg

Germany

Latvia

Denmark

Monaco

Estonia

Republic of Moldova

Spam

Madagascar

Finland

Mali

France

Mongolia
Mauritania

Gabon

Malawi

Mexico

Niger

Netherlands

Norway

New Zealand

Poland

Portugal

Romania

Russian Federation

Sudan

Sweden

Singapore

Slovenia

Slovakia

Swaziland

Chad

Togo

Tajikistan

Trinidad and Tobago

Ukraine

Uganda

United States of America

Uzbekistan

Viet Nam

WO 97/29366

PCT/AU97/00071

Enzyme Detection Biosensors

The present invention relates to biosensors and methods involving
the use of these biosensors in detecting the presence of enzymes by
5 detecting their enzymatic activity.

A number of proteins which are useful as immunodiagnostic
analytes and disease markers have the additional property of enzymatic
activity, in particular protease activity. In addition., other classes of proteins
exhihibit nuclease activity.
10 Prostate Specific Antigen (PSA), a diagnostic marker for prostate

cancer is an example of a protein which exhibits protease activity, and
belongs to the class of proteins known as the serine proteases. Examples of
other proteases which are important immunodiagnostic markers include
blood coagulation enzymes, elastase. cathepsin B.
15 There are also a number of important industrial enzvmes such as

subtilisin, papain and a-amylase.

Examples of important nucleases are restriction enzymes, e.g.,
BainHl. HindlU, polymerases which can act as nucleases under certain
conditions, e.g., T4 DNA polymerase, reverse transcriptase, which acts as an
20 Rnase under certain conditions, e.g., Rnase H, and exo- and endo-nucleases,
e.g.. Si nuclease.

Current diagnostic tests employ immunoassays for the detection of
PSA (e g. a number of analytical instruments such as Abbott's AXsym,
Boehringer Mannheim's Elecsys, and CIBA-Corning's ACS-180. all have
25 ELISA-based PSA tests). These tests use antibodies raised against the PSA
molecule which recognise the specific epitope sites within the protein
molecule.

A variation on these approaches is disclosed in International Patent
application No. PCT/AU95/00536. In this reference there is disclosed a range
30 of substrates specifically cleaved by PSA. There is also disclosure in this
reference of an assay system for proteases such as PSA which make use of
the activity of the protease. This assay system involves the use of a ligand to
capture the PSA and the subsequent use of a substrate for the PSA.

The present inventors have developed devices and methods for the
35 detection of enzymes which make use of the protein's protease activity.

These devices and methods involve the use of membrane based biosensors.

WO 97/29366

PCT/AU97/00071

Information regarding such biosensors can be found in International Patent
Application Nos PCT/AU88/00273, PCT/AU89/00352, PCT/AU90/00025,
PCT/AU92/00132, PCT/AU93/00509, PCT/AU93/00620, PCT/AU94/00202 and
PCT/AU95/00763. The disclosure of each of these applications is included
5 herein by reference.

The present invention involves providing a substrate for the enzyme
to be detected and then sensing the digestion of the substrate by the enzyme.
This may be achieved in a number of ways, for example the digestion of the
substrate may remove a group from the ionophore thereby releasing the

10 ionophore so that it (diffuses laterally within the membrane or may result in
an increase in the ability of ions to pass through the ionophore simply by a
reduction in "steric" hindrance. Alternatively the digestion of the substrate
when attached to a membrane spanning component may result in the release
of the ionophore such that it may diffuse laterally within the membrane.

15 Clearly this could also be achieved by digestion of substrates attached to
both the ionophore and membrane spanning component.

In another arrangement the digestion of the substrate results in the
release of ionophore including probe which then inserts itself into the
membrane.

20 Accordingly, in a first aspect the present invention consists in a

biosensor for use in detecting the presence of an enzyme in a sample, the
biosensor comprising a membrane and means for determining the
impedance of the membrane, the membrane having ionophores therein to
which are attached linkers, the linkers being cleavable by the enzyme to be
25 detected, the cleavage of the linker causing a change in the ability of ions to
pass through the membrane via the ionophores.

In a preferred embodiment of the present invention the linker is
attached to the membrane such that the ionophore is prevented from
diffusing laterally within the membrane. It is preferred that the linker is
30 attached to membrane spanning components provided in the membrane.
This attachment may be achieved in a number of ways such as covalent
attachment, however, it is presently preferred that the attachment is
achieved by providing on each of the linker and membrane spanning
component one member of a ligand binding pair. A preferred ligand binding
35 pair is biotin streptavidin. In another preferred arrangement both the

membrane spanning component and the linker are provided with moieties

WO 97/29366

PCT/AU97/00071

which are both bound to the same molecule, for example biotin is provided
on both the membrane spanning component and the linker and there is
cross-linking via streptavidin.

The moiety on the membrane spanning component may also be
5 attached via a linker. This may be the same linker as that provided on the
ionophore or may be different.

In a further preferred embodiment the membrane comprises a first
and second layer of a closely packed array of amphiphilic molecules, a
plurality of ionophores and a plurality of membrane-spanning lipids
10 prevented from lateral diffusion in the membrane, the ionophores

comprising first and second half membrane spanning monomers, the first
half membrane spanning monomers being provided in the first layer and the
second half membrane spanning monomers being provided in the second
layer, the first half membrane spanning monomers being prevented from
15 lateral diffusion in the first layer, the second half membrane spanning
monomers being linked to the membrane spanning lipids via the linker.
Following cleavage of the linker by the enzyme the second half membrane
spanning monomers can diffuse laterally within the second layer
independent of the first half membrane spanning monomers.
20 In a second aspect the present invention consists in a biosensor for

use in detecting the presence of an enzyme in a sample, the biosensor
comprising a membrane and means for determining the impedance of the
membrane, the membrane having a plurality of ionophores and a plurality of
membrane-spanning components therein, the membrane-spanning
25 components having attached thereto linker molecules to which are

connected the ionophores, the linker molecules being cleavable by the
enzyme to be detected, the cleavage of the linker molecules causing a
change in the ability of ions to pass through the membrane via the
ionophores.

30 In a preferred embodiment the membrane comprises a first and

second layer of a closely packed array of amphiphilic molecules and the
membrane-spanning components are prevented from lateral diffusion in the
membrane. Preferably the ionophores comprise first and second half
membrane spanning monomers, the first half membrane spanning monomers

35 being provided in the first layer and the second half membrane spanning
monomers being provided in the second layer with the first half membrane

WO 97/29366

PCT/AU97/00071

spanning monomers being prevented from lateral diffusion in the first layer.
The second half membrane spanning monomers are connected to the
membrane-spanning components via the linker molecule.

The ionophores in both these aspects are preferably gramicidin or
5 analogues thereof.

While a range of enzymes can be detected using the biosensor or the
present invention the biosensor is particularly useful in the detection of
proteases, in particular those of clinical importance such as PSA, fibrinogen
etc.

10 In a third aspect the present invention consists in a biosensor for the

detection of enzymes comprising first and second zones, means to allow
addition of a sample suspected to contain an enzyme to the first zone, the
first zone containing a probe linked to a carrier via a linker cleavable by the
enzyme and means to allow passage of unlinked probe from the first zone to

15 the second zone; the second zone including a membrane the impedance of
which is dependent on the presence or absence of the probe and means to
measure the impedance of the membrane.

In a preferred embodiment of this aspect of the present invention the
membrane comprises a first and a second layer of a closely packed array of

20 amphiphilic molecules and a plurality of ionophores comprising a first and
second half membrane spanning monomers, the first half membrane
spanning monomers being provided in the first layer and the second half
membrane spanning monomers being provided in the second layer, the
second half membrane spanning monomers being capable of lateral diffusion

25 within the second layer independent of the first half membrane spanning
monomers, the first half membrane spanning monomers being prevented
from lateral diffusion in the first layer, and a ligand provided on at least the
second half membrane spanning monomers, said ligand being reactive with
the probe or a portion thereof, the binding of the probe to the ligand causing

30 a change in the relationship between the first half membrane spanning

monomers and the second half membrane spanning monomers such that the
flow of ions across the membrane via the ionophores is allowed or
prevented.

In a preferred embodiment the probe includes streptavidin and the
35 ligand includes biotin.

WO 97/29366

PCT/AU97/00071

In yet another preferred embodiment the probe includes an
ionophore such that when the probe comes into contact with the membrane
the ionophore inserts itself into the membrane changing the impedance of
the membrane! As an example of such an arrangement the probe may
5 include valinomycin which inserts itself into the membrane.

In a preferred embodiment of the present invention the enzyme to be
detected is a protease in particular Prostate Specific Antigen. In this case it
is preferred that the linker or linker molecule includes the sequence
Ala-Val-Tyr.

10 As will be recognised by those skilled in the art the actual linker

used will depend on the enzyme to be detected. Examples of some enzymes
and their corresponding substrates are set out in Whittaker et a/. Analytical
Biochemistry: 220. 238-243 (1994), the disclosure of which is incorporated
by cross-reference.

*5 In a further aspect the present invention consists in a method of

detecting the presence of an enzyme in a sample comprising adding the
sample to the biosensor of the first or second or third aspect of the present
invention and measuring the change in impedance of the membrane.
As will be readily apparent the biosensors and methods of the
20 present invention do not detect total enzyme: they detect only active

enzyme. This is important as in a number of situations it is the amount of
active enzyme present which is of importance not simply the total amount of
enzyme present as would be measured in a standard sandwich ELISA.

It will also be apparent that the sensors of the present invention can
25 be used to detect a wide range of enzymes. These enzymes include

nucleases, protease amylases etc. The sensors are adapted to the particular
enzyme to be detected by adjusting the make-up of the linker. For example
to detect proteases the linker will typically include a peptide portion which
is cleaved by the enzyme. Information regarding peptide sequences cleaved
30 by specific proteases is provided in Whittaker et al referred to above. Where
the enzyme to be detected is a nuclease the linker will typically include a
nucleic acid sequence. Information regarding specific sequences cleaved by
specific enzymes can be found in "Current Protocols in Molecular Biology"
Ausebel et ol (1987) John Wiley & Sons, NY.
35 The sensors of the present invention may also find use in drug

development for determining DNA-drug binding sites. The sensors could

WO 97/29366 PCT/AU97/00071

also be used in determining DNA-protein binding sites. The sensors may
also find use in diagnosing infection. For example the sensors could be used
to detect enzyme activity specifically associated with a pathogen.

Industrially and clinically relevant proteases and substrates include
5 thrombin and serine proteases including PSA. A list of lysis enzymes is
found in "Specificity of Proteolysis" Borivoj Keil (1992) Springer Verlag NY
pp. 283-323. Useful ones are the serine and cysteine proteases. See also
"Proteolytic Enzymes": a Practical Approach" R.J. Benyon & J.S. Bond (eds)
1989 Oxford University Press NY p232, pp. 241-249. Commercially
10 significant proteases and protease inhibitors for which the present

technology is relevant are available in serine, cysteine, aspartic and metallo
types. The serine proteases include the endoproteinase-Arg-C, -Glu-C,
Lys-C, factor Xa, proteinase K. subtilisin and trypsin, and the exopeptidases
acylamino-acid-releasing enzyme, carboxypeptidase P, and carboxypeptidase
15 Y. The cysteine proteases include the endopeptidases bromelain, cathepsin
B, clostripain, papain, and the exopeptidases cathepsin C and pyroglutamate
aminopeptidase. The aspartic proteases include the endopeptidases
cathepsin D and pepsin. The metallo proteases include the endopeptidase
thermolysin and the exopeptidases aminopeptidase M, carboxypeptidase-A, -
20 B and leucine aminopeptidase. The listing is not intended to be exclusive
and indicates the broad utility of the present invention. Other commercially
useful proteases are listed in the publications cited above, which are
included herein by reference. For example it also includes the endopeptide
endoproteinase-Asp-N of unknown type.

In order that the nature of the present invention may be more clearly
understood preferred forms thereof will now be described by reference to the
following Examples and accompanying Figures.

Figure 1 shows a schematic representation of an embodiment of the
30 device of the third aspect of the present invention. As can be seen from this
Figure the device 10 includes a first zone 11 and a second zone 12. First
zone 11 is provided with polymer beads 13 (carrier) linked to streptavidin 14
(probe) via a peptide linker 15. The peptide linker 15 is cleavable by the
protease 16.

35 As shown in this Figure upon addition of the protease (or a nuclease)

16 the streptavidin 14 is released and passes to the second zone 12. Second

WO 97/29366

PCT/AU97/00071

zone 12 includes a biosensor membrane 17 which detects the presence of
streptavidin 14. Streptavidin 14 reaching biosensor membrane 17 causes a
change in the impedance of the membrane.

Figure 2 shows an embodiment of the first and/or second aspect of
5 the invention. As shown in Figure 2 the biosensor membrane 20 includes a
membrane 21 and electrode 22. The membrane 21 has a first layer 23 and
second layer 24 of arrays of amphiphilic molecules. Included in layer 24 is a
first half membrane-spanning monomer 25 which is prevented from lateral
diffusion within the membrane. Layer 23 includes a second half membrane-

10 spanning monomer 26. The membrane also includes a membrane-spanning
lipid 27 which is also prevented from diffusing laterally within the
membrane. The second half membrane-spanning monomer 26 is linked to
the membrane-spanning lipid 27 via a peptide 28. The peptide 28 is
cleavable by protease 29. Upon cleavage of the peptide 28 by protease 29 the

15 half membrane-spanning monomer 26 is free to diffuse laterally within the
membrane. This results in a change in impedance of the membrane.

Examples

20 Example 1:

Protease cleavage of streptavidin-gramicidin linkage

1st layer: 9.3nM Linker Gramicidin B (Fig 3)
25 l.lpM Membrane Spanner Lipid D (Fig 4)

37|iMMAAD (Fig 5)
75 mM Linker Lipid A (Fig 6)

2nd layer: lOmM (DPE-PC (Fig 7):GDPE (Fig 8) = 7:3): Biotinylated
30 Gramicidin E (Fig 9) = 66,677:1 in ethanol.

Electrodes with freshly evaporated gold (1000A) on a chrome
adhesion layer (200A on glass microscope slides) were dipped into an
ethanolic solution of the first layer components for 1 hour at room
35 temperature, rinsed with ethanol. then stored at 4°C under ethanol until
used for impedance measurements. The slide was clamped into a block

WO 97/29366

PCT/AU97/00071

containing teflon coated wells which defined the area of the working
electrode as approximately 16mm 2 .

5nL of the second layer was added to the working electrode before
addition of a 150jjL volume of phosphate buffered saline (6.26mM NaCl,
5 59.4mM NaH 2 P0 4 .2H 2 O, 2.53mM Na 2 HP0 4 . 12H 2 0, 50mM EDTA at pH 7.4;
PBS). The electrode was then washed 4 times using PBS and raised to 60°C
over a 30 minute period. Streptavidin was added to the sensor wells (5^L
O.Olmg/ml in PBS) and incubated. The binding of streptavidin to the
biotinylated gramicidin E gave a decrease in the admittance at minimum

10 phase (Figure 10). After 15 minutes the excess streptavidin was washed out
with PBS. Wells with no added streptavidin were run as controls.

Proteinase K was added to sensing and control wells to give end well
concentration at 12.5mg/ml (Boehringer Mannheim D-68298 made in PBS).
Addition of Proteinase K to control wells caused no significant change in

15 membrane admittance characteristic. Sensor membranes to which

streptavidin was bound exhibited an increase in admittance at minimum
phase (Figure 11). The amount and rate of increase of admittance at
minimum phase is related to the amount of proteinase K present in the test
solution and therefore can be used to determine enzymatic activity in test

20 solutions.

Example 2:

Dnase 1 cleavage of DNA-bound channels

1st layer: 9.3nM Linker Gramicidin B

1. luM Membrane Spanner Lipid D
27.5nM Membrane Spanner Lipid C (Fig 12)
37nMMAAP
30 75 nM Linker Lipid A

2nd layer: 14mM (DPE-PC:GDPE = 7:3): Biotinylated Gramicidin E
= 50,000:1 methanol.

35 Electrodes with freshly evaporated gold (1000 A) on a chrome

adhesion layer (200A) on glass microscope slides) were dipped into an

WO 97/29366

PCT/AU97/00071

ethanolic solution of the first layer components for 1 hour at room
temperature, rinsed with ethanol. then stored at 4°C under ethanol until
used for impedance measurements. The slide was clamped into a block
containing teflon coated wells which defined the area of the working
5 electrode as approximately 16mm 2 .

5j.iL of the second layer was added to the working electrode before
addition of a 180jiL volume of phosphate buffered saline (lOmM NaH 2 P0 4 ,
lmM KH 2 PO<, 137mM NaCl, 2.7mM KCI: PBS). The electrode was washed 4
10 times using PBS. These steps were carried out at room temperature. All the
subsequent steps were carried out at 30 a C. Streptavidin was added to all the
wells (5^L O.Olmg/ml in PBS) and allowed to react with biotinylated
gramicidin E for 10-15 minutes before washing out excess unbound
streptavidin with PBS, 5^L of a 1:1 mixture of DNA probe F (200nM): DNA
15 probe G (200nM in PBS) was added to the sensor wells. A DNA non-specific
binding probe H {5\iL 400 nM in PBS) was added to control wells. Binding
probe H is non-complementary to the target DNA of interest and hence target
DNA should not bind. The probes were allowed to react with streptavidin
for 10-15 minutes then excess unbound probes were washed out with PBS.
20 100 uL of DNA target I (lOnM) in PBS was added to each well. The binding
of DNA target I to the sensor wells gave a decrease in the admittance at
minimum phase, but no significant change in membrane admittance in
control wells (Figure 13). After 15 minutes unbound DNA target I was
washed out with DNase 1 activation buffer. DNase 1 activation buffer
25 consists of 50nM Tris. HC1, pH 7.6, 50nM NaCl, lOnM MgCl 2 . lOnM MnCl 2 ,
0.2 mg/mL BSA. DNase 1 was added (2pL lmg/mL in a 50%w/v glycerol
solution of 20mMTris.HCl, pH 7.6. lmM MgCl 2 ) to sensor and control wells.
Addition of DNase 1 gave an increase in admittance at minimum phase for
sensor wells, but no significant change for control wells (Figure 14). The
30 amount and rate of increase of admittance at minimum phase is related to
the amount of DNase 1 present in the test solution and therefore can be used
to determine enzymatic activity in test solutions.

WO 97/293*6

PCT/AU97/00071

DNA probe F:

5'biotinylated listeria probe DNA with a 31-atom phosphoramidite
linker group between the biotin and DNA.

5 5 '-bio-L-M-ATAGTTTTATGGGATTAGC-3'

DNA probe G:

5 f biotinylated cholera toxin probe DNA with a 13-atom
phosphoramidite linker group between the biotin and DNA.

5-bio-L-CTCCGGAGCATAGAGCTTGGAGG-3'

DNA non-specific binding probe H:

5 ? biotinylated 15-mer oligonucleotide with a 31-atom
15 phosphoramidite linker group between the biotin and DNA, which is

non-complementary to all parts of the target DNA sequence.

5 -bio-L-M-ATTGCTACGTATACG-3 f

20 DNA target I:

52 base DNA sequence containing the 19-base listeria sequence, a 10
base 'spacer and the 23 base cholera toxin sequence.

5- GCTAATCCCATAAAACTAT GC^^^

WO 97/29366

where:

PCT/AU97/00071

bio = biotin

O-DMT

O
I

~o—p=o
I

It will be appreciated by persons skilled in the art that numerous
variations and/or modifications may be made to the invention as shown in
the specific embodiments without departing from the spirit or scope of the
invention as broadly described. The present embodiments are. therefore, to
be considered in all respects as illustrative and not restrictive.

WO 97/29366

PCT/AU97/00071

CLAIMS:-

1. A biosensor for use in detecting the presence of an enzyme or
enzymes in a sample, the biosensor comprising a membrane and means for
determining the impedance of the membrane, the membrane having

5 ionophores therein to which are attached linkers, the linkers being cleavable
by the enzyme or enzymes to be detected, the cleavage of the linker causing
a change in the ability of ions to pass through the membrane via the
ionophores.

2. A biosensor as claimed in claim 1 in which the linker is attached to
10 the membrane such that the ionophore is prevented from diffusing laterally

within the membrane.

3. A biosensor as claimed in claim 2 in which the linker is attached to
membrane spanning components provided in the membrane.

4. A biosensor as claimed in claim 3 in which the linker is attached to
15 the membrane spanning component via a ligand binding pair.

5. A biosensor as claimed in any one of claims 1 to 4 in which the
membrane comprises a first and second layer of a closely packed array of
amphiphilic molecules, a plurality of ionophores and a plurality of
membrane-spanning lipids prevented from lateral diffusion in the

20 membrane, the ionophores comprising first and second half membrane
spanning monomers, the first half membrane spanning monomers being
provided in the first layer and the second half membrane spanning
monomers being provided in the second layer, the first half membrane
spanning monomers being prevented from lateral diffusion in the first layer,

25 the second half membrane spanning monomers being linked to the
membrane spanning lipids via the linker.

6. A biosensor as claimed in any one of claims 1 to 5 in which the
ionophores are gramicidin or analogues thereof.

7. A biosensor as claimed in any one of claims 1 to 6 in which the
30 enzyme to be detected is a protease.

8. A biosensor as claimed in claim 7 in which the protease is PSA.

9. A biosensor as claimed in any one of claims 1 to 6 in which the
enzyme to be detected is a nuclease.

10. A biosensor for use in detecting the presence of an enzyme in a
35 sample, the biosensor comprising a membrane and means for determining

the impedance of the membrane, the membrane having a plurality of

WO 97/29366

PCT/AU97/00071

ionophores and a plurality of membrane-spanning components therein, the
membrane-spanning components having attached thereto linker molecules
to which are connected the ionophores, the linker molecules being cleavable
by the enzyme to be detected, the cleavage of the linker molecules causing a
5 change in the ability of ions to pass through the membrane via the
ionophores.

11. A biosensor as claimed in claim 10 in which the membrane
comprises a first and second layer of a closely packed array of amphiphilic
molecules and the membrane-spanning components are prevented from

10 lateral diffusion in the membrane.

12. A biosensor as claimed in claim 10 or claim 11 in which the
ionophores comprise first and second half membrane spanning monomers,
the first half membrane spanning monomers being provided in the first laver
and the second half membrane spanning monomers being provided in the

15 second layer with the first half membrane spanning monomers being
prevented from lateral diffusion in the first layer.

13. A biosensor as claimed in any one of claims 10 to 12 in which the
ionophores are gramicidin or analogues thereof.

14. A biosensor as claimed in any one of claims 10 to 13 in which the
20 enzyme to be detected is a protease.

15. A biosensor as claimed in claim 14 in which the protease is PSA.

16. A biosensor as claimed in any one of claims 10 to 13 in which the
enzyme to be detected is a protease.

17. A biosensor for the detection of enzymes comprising first and second
25 zones, means to allow addition of a sample suspected to contain a protease

to the first zone, the first zone containing a probe linked to a carrier via a
linker cleavable by the enzyme and means to allow passage of unlinked
probe from the first zone to the second zone: the second zone including a
membrane the impedance of which is dependent on the presence or absence
30 of the probe and means to measure the impedance of the membrane.

18. A biosensor as claimed in claim 17 in which the membrane
comprises a first and second layer of a closely packed array of amphiphilic
molecules and a plurality of ionophores comprising first and second half
membrane spanning monomers, the first half membrane spanning monomers

35 being provided in the first layer and the second half membrane spanning
monomers being provided in the second layer, the second half membrane

WO 97/29366

PCIYAU97/00071

spanning monomers being capable of lateral diffusion within the second
layer independent of the first half membrane spanning monomers, the first
half membrane spanning monomers being prevented from lateral diffusion
in the first layer, and a ligand provided on at least the second half membrane
5 spanning monomers, said ligand being reactive with the probe or a portion
thereof, the binding of the probe to the ligand causing a change in the
relationship between the first half membrane spanning monomers and the
second half membrane spanning monomers such that the flow of ions across
the membrane via the ionophores is allowed or prevented.
10 19. A biosensor as claimed in claim 17 or claim 18 in which the enzymes
to be detected are proteases.

20. A biosensor as claimed in claim 19 in which the protease is PSA.

21. A biosensor as claimed in claim 17 or claim 18 in which the enzyme
to be detected is a nuclease.

15 22. A biosensor as claimed in any one of claims 17 to 21 in which the
half membrane spanning monomers are gramicidin or analogues thereof.

23. A biosensor as claimed in claim 17 in which the probe includes an
ionphore.

24. A method of detecting the presence of an enzyme in a sample
20 comprising adding the sample to the biosensor as claimed in any one of

claims 1 to 23 and measuring the change in impedance of the membrane.

25. A method as claimed in claim 24 in which the enzymes to be
detected are proteases.

26. A method as claimed in claim 25 in which the protease is PSA.

WO 97/29366

PCT/AU97/00071

1/13

WO 97/29366

PCT/AU97/00071

3/13

WO 97/29366

PCT/AU97/00071

Figure 4

WO 97/29366

PCT/AU97/00071

Figure 5

WO 97/29366

PCT/AU97/00071

6/13

Figure 6

WO 97/29366

7/13

PCT/AU97/00071

GDPE

FIGURE 7

Figure 8

WO 97/29366

PCT/AU97/00071

Figure 9

WO 97/29366

PCT/AU97/00071

15 -I — : — —i : — — i : .

1500 2000 2500 3000

Time (■)

WO 97/29366

PCT/AU97/00071

WO 97/29366

PCT/AU97/00071

11/13

Figure 12

WO 97/29366

PCT/AU97/00071

12/13

Figure 13

53000 T

j~— Active cell

J Control cell

"Difference

33000-1 : : f- f_ , f

0 500 1000 1500 2000 2500

Time (s)

WO 97/29366

PCT/AU97/00071

13/13

Figure 14

50000 1

\
i
\

2500 2700 2900 3100 3300 3500 3700 3900

Time (sec)

INTERNATIONAL SEARCH REPORT

International Application No.
PCT/AU 97/00071

A. CLASSIFICATION OF SUBJECT MATTER

Int CP* G01N 27/327, 27/333, 33/50, 33/574, 33/68, 33/96

According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)
IPC : G01N 33/-, G01N 27/-

Docun^tation searched other than minimum documentation to the extent that such documents are included in the fields searched
AU : IPC as above

b ^ consu,ted dun *S * c international search (name of data base and, where practicable, search terms used)
DERWENT : Biosensor, lonophore, Ion Channel

DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

P,A

WO,A, 96/12957 (P1TTNER, F. & SCHALKHAMMER, T.) 2 May 1996.
The whole document

AU,A, 38643/95 (AUSTRALIAN MEMBRANE AND BIOTECHNOLOGY
RESEARCH INSTITUTE etal) 6 June 1 996.
Page 2 lines 18 to 34

AU,A, 56403/96 (AUSTRALIAN MEMBRANE AND BIOTECHNOLOGY
RESEARCH INSTITUTE etaj) 29 November 1996.
Page 2 lines 18 to 34

1-26

1-6, 10-13, 17, 18
22-24

1-26

Further documents arc listed in the continuation of Box C

See patent family annex

"A"

Special categories of cited documents:

document defining the general state of the art which is
not considered to be of particular relevance
earlier document but published on or after the
international filing date

document which may throw doubts on priority claim(s)
or which is cited to establish the publication date of
another citation or other special reason (as specified)
document referring to an oral disclosure, use,
exhibition or other means
document published prior to the international filing

ay*

lata document published after the intenuttional filing date or
priority date and not in conflict with the application but cited to
understand the principle or theory underlying the invention
document of particular relevance; the claimed invention cannot
be considered novel or cannot be considered to involve an
inventive step when the document is taken alone
document of particular relevance; the claimed invention cannot
be considered to involve an inventive step when the document is
combined with one or more other such documents, such
combination being obvious to a person skilled in the art
document member of the same patent family

Date of the actual completion of the international search
21 May 1997

Date of mailing of the international search report I

29 MAY <eo7

Name and mailing address of the ISA/AU

AUSTRALIAN INDUSTRIAL PROPERTY ORGANISATION

PO BOX 200

WODEN ACT 2606

AUSTRALIA Facsimile No.: (06) 285 3929

Authorized officer

D.A.LALLY ^^'^W

Telephone No.: (06) 283 2533 / J

Form PCT/ISA/210 (second sheet) (July 1992) cophin

INTERNATIONAL SEARCH REPORT

International Application Mo.
PCT/AU 97/00071

C (Continuation)

Category*

DOCUMENTS CONSIDERED TO BE RELEVANT

Citation of document, with indication, where appropriate, of the relevant passages

WO,A, 95/16206 (BIOSYSTEMS TECHNOLOGY (CORP)) 15 June 1995. ~
Page 12, lines 7 to 12

WOA 95/08637 (UNIVERSITY OF WASHINGTON STATE RESEARCH FOUNDATION)
30 March 1995

US,A, 5368712 (SYNPORIN TECHNOLOGIES, INC) 29 November 1994.
Page 5 line 11 to 16

AU.A, 65327/94 (AUSTRALIAN MEMBRANE AND BIOTECHNOLOGY RESEARCH
INSTITUTE et_al) 8 November 1994. .
Page 2 lines 18 to 34

AU,A, 56188/94 (AUSTRALIAN MEMBRANE AND BIOTECHNOLOGY RESEARCH
INSTITUTE etaD 22 June 1994.
Page 1 line 25 to page 2 line 6

AUA 51444/93 (AUSTRALIAN MEMBRANE AND BIOTECHNOLOGY RESEARCH
INSTITUTE elal) 26 April 1 994.
Page 4 line 2 to page 29 line 35.

EPA 342382 (GENERAL ELECTRIC COMPANY) 23 November 1989.
Columns 4 to 6.

GB,A, 2195450 (UNITED KINGDOM ATOMIC ENERGY AUTHORITY) 7 April 1988.
Lines 59 to 78

Relevant to
claim No.

1. 17
1-26
1-26

1-26

1-26
1-26
1-26

Form PCT/ISA/210 (second sheet) (July 1992) cophin

INTERNATIONAL SEARCH REPORT

Information on patent family members

International Application No.
PCT/AU 97/00071

This Annex lists the known "A" publication level patent family members relating to the patent documents cited
in the above-mentioned international search report. The Australian Patent Office is in no way liable for these
particulars which are merely given for the purpose of information.

Patent Document Cited in Search

Patent Family Member

Report

96/12957

AX
At

iy /vi/y4

734528

9612957

38643/95

9615454

56403/96

9636871

95/16206

AIT

13008/95

1137316

733211

5468648

9516208

21852/92

2103052

586595

935244

6508215

9221977

5607863

64970/94

100104

9406755

2158570

1124524

692097

954591

9502835

73379

8508569

953872

263754

310953

1227/95

9423300

5368712

5516890

5368712

65327/96

65327/94

9424562

695425

8509807

2161084

8294768

56188/94

2150915

672251

8504943

5591647

9412875

51444/93

9407593

670751

8505123

342382

89719

2024548

892020

4920047

2195450

261887

8622788

8721607

END OF ANNEX

Form PCT/ISA/210 (extra sheet) (July 1 992) cophin

Internet Archive Audio

Featured

Top

Images

Featured

Top

Software

Featured

Top

Books

Featured

Top

Video

Featured

Top

Mobile Apps

Browser Extensions

Archive-It Subscription

Save Page Now

Full text of "USPTO Patents Application 10687850"

See other formats